Cervical cancer is the second-most common cancer in women worldwide. In the United States, approximately 10,000 women are diagnosed each year, of which 30% are expected to die. Worldwide, nearly half a million cases are diagnosed annually, with a mortality rate of approximately 50%. In addition, HPV is associated with approximately 20% of head and neck cancers in the US, as well as with some skin and colorectal carcinomas. Although vaccines have recently become available, they are protective rather than prophylactic, and are not appropriate for use in the immunocompromised population (i.e., HIV-infected individuals). For this reason, the development of novel and effective therapeutic approaches remains an urgent need. Results from our laboratory and others have identified the E6 oncoprotein of high-risk strains of HPV as an important contributor to the oncogenicity of HPV, as it blocks multiple apoptotic pathways. This allows the virus to persist in the host and increases the probability that it will integrate into the genome and facilitate tumor formation. Our laboratory has found that E6 modifies the flow of apoptotic pathways triggered by TNF, Fas and TRAIL, in part by binding to both FADD and procaspase 8 and accelerating their degradation. We utilized mutational and peptide-based approaches to map the E6 binding site on FADD, and found that it represents a novel E6 binding motif that also appears in procaspase 8. This domain does not overlap with other known binding sites for E6, and thus functions as a motif that specifically contributes to the ability of HPV to thwart host-mediated apoptotic mechanisms. The proposed work is innovative, in that to the best of our knowledge, no other published or ongoing work is focused on the reactivation of death receptor-mediated apoptotic pathways disrupted by E6. We have already developed, optimized and tested a bead-based screening assay that can identify small molecules capable of disrupting the binding between E6 and its cellular partners. Our first specific aim focuses on further optimization of our assay, while the second specific aim focuses on configuring this assay for high throughput screening. The overall goal of our research is to identify one or more lead molecules, selected on the basis of their ability to interfere with E6/host protein interactions, which can be developed into a novel and effective therapy for HPV-associated malignancies. The goal of this specific proposal is to further develop and validate our existing bead-based assay to the point that it is ready for submission to the MLPCN. Success in achieving our stated aims will lead initially to the development of pharmacological tools for research in the HPV field, with the ultimate objective of developing novel and effective therapeutic approaches that change and improve treatment protocols for patients with established HPV-related malignancies and lesions. PUBLIC HEALTH RELEVANCE: High-risk strains of human papillomaviruses (HPV) are best known for their ability to cause cervical and head and neck cancer, though they also cause sores and warts that do not lead to cancer. The work described in this grant application is focused on finding molecules that could be turned into drugs that would be effective therapeutic treatments for HPV-associated cancers.